Cell penetrating peptides for intracellular delivery of molecules

ABSTRACT

The present invention concerns cell-penetrating peptides which comprise an amino acid sequence consisting of GLX 9 WRAX 9 WRX 1 LX 2 RSLX 9 WX 3 X 4 X 5 X 6 X 7 X 8 (SEQ ID No: 1), wherein X 1  is A, L or G, X 2  is W or none, X 3  is R or K, X 4  is K, L or S, X 5  is L or K, X 6  is R or W, X 7  is K or S, and X 8  is A, V or Q, and X 9  is W, F or Y. These CPPs can be used as vectors for delivering nucleic acids and/or proteins and/or peptides to cells, in vitro or in vivo.

The present invention pertains to the field of intracellular delivery ofmolecules such as proteins or nucleic acids. In particular, theinvention relates to a new cell-penetrating peptide (CPP) family, whichexhibits high efficiency and low toxicity.

Cellular internalization of large hydrophilic therapeutic agents such asproteins or nucleic acids is still a challenging task because of thepresence of the plasma membrane, which constitutes an impermeablebarrier for such molecules. In order to circumvent this problem, severalmethods of carrier-mediated delivery systems have been developed. Amongthem, much attention has recently been given to the use of peptide-baseddelivery systems. The use of peptides with cell-penetrating propertieshas several advantages, which are mainly due to the variousmodifications that can be done to the peptide sequence. This allows theengineering of carriers addressing different cellular subdomains and/orable to transport various types of cargoes.

Many CPPs were designed from sequences of membrane-interacting proteinssuch as fusion proteins, signal peptides, transmembrane domains andantimicrobial peptides (Morris, Chaloin et al. 2000; Jarver and Langel2004; El-Andaloussi, Holm et al. 2005). Within these sequences, shortsequences called Protein Transduction Domains or PTDs proved toefficiently cross biological membranes without the need of a carrier orof a receptor, and to deliver peptides or proteins into intracellularcompartments. Many investigations suggested that the use of PTD-basedpeptides could be of major importance for therapies against viraldiseases or cancers. Among these, the third helix of the homeodomain ofantennapedia called penetratin (Joliot and Prochiantz 2004), the Tatpeptide derived from the transactivating protein Tat of HIV-1 (Wadia andDowdy 2002), transportan (Pooga, Hallbrink et al. 1998) and VP22(Elliott and O'Hare 1997) were used to improve the cellular uptake ofpeptides, proteins or oligonucleotides.

A second category of cell-penetrating peptides, designated asamphipathic peptides, has been described. An amphipathic molecule can bedefined, in short, as consisting of two domains: a hydrophilic (polar)domain and a hydrophobic (non-polar) domain. For peptides, theamphipathic character can arise either from the primary structure, orfrom the secondary structure. Primary amphipathic peptides can bedefined as the sequential assembly of a domain of hydrophobic residueswith a domain of hydrophilic residues. Secondary amphipathic peptidesare generated by the conformational state which allows the positioningof the hydrophobic and hydrophilic residues on opposite sides of themolecule.

Other peptides, such as polyarginine-based peptides, calcitonin-derivedpeptides, and oligomers, have also been proposed as tools forintracellular delivery of therapeutics.

Deshayes et al. have reviewed the available data concerning the use ofthe above-mentioned cell-penetrating peptides for delivering moleculesinto cells (Deshayes, Morris et al. 2005). It appears that the currentvectors are limited, because of their lack of efficiency and/or theirtoxicity, and that little is known about the pathway of their cellularuptake, which constitutes a handicap for improving their efficiency.

The present invention pertains to a new family of cell-penetratingpeptides having particularly advantageous properties.

A first aspect of the present invention is hence a cell-penetratingpeptide which comprises an amino acid sequence consisting ofGLX₉RAX₉RX₁LX₂RSLX₉X₃X₄X₅X₆X₇X₈ (SEQ ID No: 1), wherein X₁ is A, L or G,X₂ is W or none, X₃ is R or K, X₄ is K, L or S, X₅ is L or K, X₆ is R orW, X₇ is K or S, X₈ is A, V or Q, and X₉ is W, F or Y. According to theinvention, X₉ can also be another aromatic chemical group. In apreferred embodiment of the invention, X₉ is W, which means that thecell-penetrating peptide comprises an amino acid sequence consisting ofGLWRAWRX₁LX₂RSLWX₃X₄X₅X₆X₇X₈ (SEQ ID No: 25), with X₁ to X₈ as describedabove. The CPPs according to the invention have a secondary amphipathicstructure. In the CPPs according to the invention, the amino acids canbe either L- or D-amino acids.

In the present text, a “cell-penetrating peptide”, also called a“peptide carrier”, is a molecule, the core of which is a peptide. Otherchemical groups can however be covalently bound to said peptidic core,in order to improve the overall stability of the molecule, and/or toprovide it with additional properties, such as targeting ability. Forexample, a cell-penetrating peptide according to the invention canfurther comprise, covalently linked to the C-terminal extremity of thepeptidic core of SEQ ID No: 1, one or several groups chosen amongst acysteamide, a cysteine, a thiol, an amide, a carboxyl, a linear orramified C₁-C₆ alkyl optionally substituted, a primary or secondaryamine, an osidic derivative, a lipid, a phospholipid, a fatty acid, acholesterol, a poly-ethylene glycol, a nuclear localization signal(NLS), and/or a targeting molecule. Alternatively or additionally, thecell-penetrating peptide of the invention can also comprise, covalentlylinked to the N-terminal end of the peptidic core of SEQ ID No: 1, oneor several chemical entities chosen amongst an acetyl, a fatty acid, acholesterol, a poly-ethylene glycol, a nuclear localization signal,and/or a targeting molecule. If necessary, for example in the case ofN-terminal addition of cholesterol, a peptidic bridge can be used tobind a non-peptidic molecule to the peptidic core of the CPP. An exampleof such a bridge is —CA_(β)—.

Although some of the entities which can be covalently bound to thepeptide of SEQ ID No: 1 comprise amino acids, the invention does notencompass every peptide or protein comprising SEQ ID No: 1, whatever theamino acids surrounding the core sequence of SEQ ID No: 1. Indeed, theonly additional amino acid sequences that can be bound to a CPPaccording to the invention are the following:

(i) a peptidic bridge such as CAβ: the size of such a bridge will neverexceed 5 amino acids. Preferably, the bridge consists of 1, 2 or 3 aminoacids.

(ii) a nuclear localization signal (NLS): such signals are well known inthe scientific literature. An example of NLS that can be used accordingto the present invention is PKKKRKV (SEQ ID No: 27). Other NLS which canbe used can contain up to 11 amino acid residues, in the case of abipartite NLS (Morris, Chaloin et al. 2002).

(iii) targeting peptides: targeting molecules which can advantageouslybe bound to the CPPs according to the invention, can be of peptidicnature. Examples of targeting peptides are epitopes, antibody fragments(Fab) which specifically target tissues or cell surface components orreceptors, and the like. Whatever the molecules bound to the peptidiccore of SEQ ID No: 1, CPPs according to the invention must retain theirvector efficiency, i.e., their ability (i) to interact with the cargo,and (ii) to deliver said cargo into the cells.

Therefore, in the context of the present invention, the sequence of atargeting peptide will preferably be no longer than 15 amino acids. Aexamples of such peptides are the RGD motif, NGR, transferrine, antibodyfragments, and any of the peptides listed in Table 1 of the review byAllen (Allen 2002).

Apart from the targeting peptides, other targeting molecules can bebound to the CPPs of the invention, in order to obtain a specificdelivery of molecules to particular cell types and/or organs. Forexample, sugars, such as monosaccharides (ex glucose, galactose,glucosamine ou galactosamine), oligosaccharides, polysaccharides, ortheir analogues, as well as some oligonucleotides, or some organicmolecules such as folate, can be used to that aim. Their targetingactivity is due to the fact that they are recognized as ligands by somereceptors which are over-expressed at the surface of cells in the zoneof interest.

In a particular embodiment of the cell-penetrating peptide according tothe invention, the amino acid sequence of the peptidic core is chosen inthe group consisting of

GLWRALWRLLRSLWRLLWKA; (SEQ ID No: 2) GLWRALWRALWRSLWKLKRKV; (SEQ ID No:3) GLWRALWRALRSLWKLKRKV; (SEQ ID No: 4) GLWRALWRGLRSLWKLKRKV; (SEQ IDNo: 5) GLWRALWRGLRSLWKKKRKV; (SEQ ID No: 6) GLWRALWRLLRSLWRLLWKA; (SEQID No: 7) GLWRALWRALWRSLWKLKWKV; (SEQ ID No: 8) GLWRALWRALWRSLWKSKRKV;(SEQ ID No: 9) GLWRALWRALWRSLWKKKRKV; (SEQ ID No: 10) andGLWRALWRLLRSLWRLLWSQ. (SEQ ID No: 11)

Preferred cell-penetrating peptides according to the invention areCADY-1 (Ac-GLWRALWRLLRSLWRLLWKA-Cya) and CADY-2(Ac-GLWRALWRALWRSLWKLKWKV-Cya).

Another aspect of the present invention is a complex comprising acell-penetrating peptide as described above, and a cargo selectedamongst nucleic acids, peptides, proteins, contrast agents, and toxins.

In a particular embodiment of the complexes of the invention, the cargois a siRNA selected to silence a target MRNA. In this embodiment, theamino acid sequence of (core of) the cell-penetrating peptide preferablyis GLWRAWRX₁LX₂RSLWX₃X₄X₅KX₆ (SEQ ID No: 26), wherein X₁ is A, L or G,X₂ is W or none, X₃ is R or K, X₄ is K or L, X₅ is R or W, X₆ is A or V.For example, the amino acid sequence of the cell-penetrating peptide ischosen in the group consisting of:

GLWRALWRLLRSLWRLLWKA; (SEQ ID No: 2) GLWRALWRALWRSLWKLKRKV; (SEQ ID No:3) GLWRALWRALRSLWKLKRKV; (SEQ ID No: 4) GLWRALWRGLRSLWKLKRKV; (SEQ IDNo: 5) GLWRALWRGLRSLWKKKRKV; (SEQ ID No: 6) and GLWRALWRGLRSLWKKKRKV.(SEQ ID No: 7)

According to the invention, preferred CPPs for siRNA delivery have anamino acid sequence corresponding to SEQ ID Nos 2, 6 or 7.

In another embodiment of the complexes of the invention, the cargo is apeptide or a protein. In this embodiment, the amino acid sequence of(core of) the cell-penetrating peptide is SEQ ID No: 1, as describedabove, preferably with the proviso that X₁ is not G and X₈ is not A.Examples of amino acid sequences of cell-penetrating peptides that canbe incorporated in complexes with peptides or proteins are thefollowing:

GLWRALWRALWRSLWKLKWKV (SEQ ID No: 8) GLWRALWRALWRSLWKSKRKV (SEQ ID No:9) GLWRALWRALWRSLWKKKRKV (SEQ ID No: 10) GLWRALWRALWRSLWKLKRKV (SEQ IDNo: 3) GLWRALWRLLRSLWRLLWSQ (SEQ ID No: 11) GLWRALWRLLRSLWRLLWSQPKKKRKV(SEQ ID No: 12)

According to the invention, preferred CPPs for proteins and peptidedelivery have an amino acid sequence corresponding to SEQ ID Nos 8, 9 or10. Depending on the cargo and the application, the skilled artisan canuse the intracellular targeting properties of those CPPs, especiallythose of SEQ ID Nos 8 and 9, which target the nucleus and the cytoplasm,respectively.

Of course, the cell-penetrating peptides comprised in the complexesaccording to the invention can be modified as mentioned above, bybinding additional molecules to the N-terminal and/or the C-terminalends of their peptidic core of SEQ ID No: 1. In a preferred embodimentof such complexes, the cell-penetrating peptide comprises an acetylgroup covalently linked to its N-terminus, and/or a cysteamide groupcovalently linked to its C-terminus. Alternatively or additionally, thecell-penetrating peptide further comprises a cholesterol molecule,covalently linked to its C-terminus or its N-terminus. When bound to theN-terminus, the cholesterol molecule can be linked via a bridge asdescribed above, for example a CA_(β) bridge.

The inventors have investigated various parameters in order to optimizethe cargo delivery, and have found that when the size of the complexesis <500 nm, the mechanism of cellular uptake of said complexes isindependent on the endosomal pathway. Hence, the size of the complexesaccording to the invention is preferably between 50 and 300 nm and, morepreferably, between 100 and 200 nm. This corresponds, for siRNA/CADYcomplexes, to a ratio of 1/20 to 1/25.

According to an advantageous embodiment of the complex according to theinvention, at least part of the cell-penetrating peptides are bound to atargeting molecule. As mentioned above, a targeting molecule can be apeptide, a sugar, etc. When only part of the CPPs are initially bound toa targeting molecule, this latter can be chosen bigger than the maximalsize of targeting molecules which are acceptable when all the CPPs arebound to targeting molecules. The targeting molecule can be eithercovalently linked to a CPP, or bound to the complex via non-covalentbounds. In some cases, the cargo itself can be a targeting molecule,especially for targeting tumor cells.

A therapeutic composition comprising a complex as described above is, ofcourse, part of the present invention.

In one particular aspect, the invention pertains to the use of acell-penetrating peptide, or of a complex as described above, for thepreparation of a therapeutic composition for use in anticancer therapy.For example, a therapeutic composition according to the inventioncomprises anti-cyclin B 1 siRNA/CADY complexes, and/or p1p27/CADYcomplexes, and/or pRXL/CADY complexes.

The invention also pertains to a method for delivering a molecule into acell in vitro, comprising a step of putting said cell into contact witha complex comprising said molecule and cell-penetrating peptides asdescribed above.

The invention is further illustrated by the following figures andexamples.

LEGENDS TO THE FIGURES

FIG. 1: Schematic representation of CADY cellular uptake mechanism.

FIG. 2: Structure of CADY in the interaction with the membrane.

(A) modelisation

(B) CD experiments—Far UV CD spectra were recorded on a JASCO J-810spectro-polarimeter (JASCO Corporation, Tokyo, Japan) using a quartzcuvette with a path length of 1 mm. Spectra were corrected from thebaseline buffer spectrum and smoothed with the JASCO software. Peptideswere dissolved to a fixed concentration of 50 μM in water or in PBS. CDspectra were collected at 23° C. and each spectrum represents theaverage of 4 scans. CADY-1 & CADY-2 adopt a α-helical structurecharacterized by two minima at 208 nm and 222 nm, respectively.

FIG. 3: Formation of CADY/cargo complexes.

Binding of cargoes to CADY was monitored by steady state fluorescencespectroscopy. The intrinsic fluorescence associated to the Trp residuesof CADY was used as sensitive probe. The fluorescence of CADY iscentered at 340 nm upon excitation at 290 nm. Experiments were performedin phosphate buffer (PBS). A fixed concentration of CADY (0.1 μM) wastitrated by increasing concentrations of cargoes.

(A) For the experiments with CADY-1, two different siRNAs (doublestranded RNA) targeting cyclin B 1 and GAPDH, were used.

(B) For the experiment with CADY-2, a short 17-residues peptide wasused.

FIG. 4: Toxicity of CADY peptides.

Cells were incubated in the presence of increasing concentrations (from0.1 μM to 1 mM) of CADY-1, either alone (A) or in complexes with a cargosiRNA at a ratio of 1/20 (B). Cell viability was estimated using MTTassay, after 12 hrs of incubation.

FIG. 5: CADY-1-mediated delivery of siRNA.

The different cell lines were cultured as described in the ATCCprotocol. Cell lines were cultured in Dulbecco's Modified Eagle's Mediumsupplemented with 2 mM glutamine, 1% antibiotics (streptomycin 10000μg/ml, penicillin, 10000 IU/ ml) and 10% (w/v) fetal calf serum (FCS),at 37° C. in a humidified atmosphere containing 5% CO₂. CADY/siRNAcomplexes were formed by incubation of siRNA (50 nM) with CADY at amolecular ratio of 1:20 (i.e., one molecule of siRNA for 20 molecules ofCADY), in 500 μl of DMEM for 30 min at 37° C. Cells grown to 40 to 60%confluence were then overlaid with preformed complexes. After 30 minincubation at 37° C., 1 ml of fresh DMEM supplemented with 10% fetalcalf serum was added directly to the cells, without removing the overlayof CADY/siRNA complexes, and cells were returned to the incubator.Control experiments were performed with siRNA (50 nM) transfected withcationic lipids Oligofectamine (Invitrogen, Carlsbad, US), according tothe guidelines of the manufacturer. A well established siRNA targetingGAPDH protein (Ambion-CA) was used as cargo. The knockdown of the GAPDHat the protein level was quantified 48 hrs after transfection by Westernblot analysis using polyclonal antibody (Ref. ABCAM GAPDHantibody—ab9385 rabbit polyclonal). The percentage of GAPDH knockdownwas estimated by comparison with control essays using a mismatch siRNAdelivered with CADY at the same molecular ratio. Data are the average of4 separated experiments.

FIG. 6: Mechanism of cellular uptake of CADY.

(A) impact of both the size of the particle and of the molar ratioCADY/siRNA on the efficiency of the siRNA. CADY/siRNA-GAPDH complexeswere formed by incubation of siRNA (50 nM) with increasing molecularratio of CADY from 5:1 to 50:1. Experiments were performed on HumanHS-68 fibroblasts or Hela cell lines. The size of the particle wasdetermined by light scattering and the knock down of GAPDH protein wasquantified by Western blot analysis.

(B) role of the endosomal pathway on the efficiency of CADY-mediatedsiRNA delivery. CADY/siRNA-GAPDH complexes were formed by incubation ofa fixed concentration of siRNA (50 nM) with CADY-1 at two molar ratios:20:1 and 50:1, which correspond to CADY/siRNA-GAPDH particles of 100-200nm and of 500 nm diameter size, respectively. In order to determine thecellular uptake mechanism of CADY, the transfection experiments wereperformed in the presence of different inhibitors of the endosomalpathway, as previously described by Dowdy and colleagues (Wadia, Stan etal. 2004). The following inhibitors of the endosomal pathway were used:Heparin (20 μg/ml), Nystatin or Filipin (25 μg/ml), Cytochalasin D (5μM). The cells were incubated in the presence of the inhibitor one hourprior transfection; the inhibitor was then maintained in the culturemedium for one hour after transfection. Cells were then extensivelywashed, trypsine was added in order to remove any CADY/siRNA complexassociated to the cell membrane. Cells were the cultured for 24additional hours. Experiments were performed on HS-68 fibroblasts orHela cell lines. The knock down of GAPDH protein was quantified byWestern blot analysis.

FIG. 7: CADY-mediated cyclin-BI siRNA delivery

The effect of increasing concentrations of siRNA from 2 nM to 100 nMcomplexed with CADY (ratio 1/20) on cyclin B1 protein levels wasanalyzed in several cell lines (Hela cells, human fibroblasts (HS 68)and 293 cells). A stock solution of CADY/siRNA formulation was preparedusing a siRNA concentration of 100 nM associated with a CADY-1 at amolar ratio of 1/20. Lower concentrations of formulated siRNA (from 50nM to 0.5 nM) were obtained by serial dilution of the stock solution inPBS, in order to maintain the siRNA/CADY ratio, and therefore the sizeof the particle around 200 nm diameter. SiRNA/CADY complexes wereoverlaid onto asynchronous cultured cells in the presence of FBS (10%).

(A) The effect of Cyclin B1 siRNA was monitored on cell cycleprogression by FACS analysis 30 hrs after transduction.

(B) Cyclin B1 protein levels were quantified by Western blotting 30 hrsafter transduction. To that aim, cells were collected and proteins wereseparated on SDS gel (12%), then transferred onto nitro-cellulose forWestern blotting. Mouse monoclonal anti-Cyclin B1 antibodies (SC-245)and rabbit polyclonal anti-Cdk2 antibodies (SC-163) for Western blottingwere obtained from Santa Cruz Biotechnology Inc., (Santa cruz, Calif.).Cdk2 protein kinase was used as a control to normalize protein levels.

FIG. 8: potency of CADY/siRNA complex to block cancer cellproliferation.

Experiments were performed on MCF-7 or U20S cells. Cells were treated onday 1 with different concentrations of siRNA and CADY/siRNA complexes(0.1 to 50 nM). A stock solution of CADY/siRNA formulation was preparedusing a siRNA concentration of 100 nM associated with a CADY-1 at amolar ratio of 1:20. Lower concentrations of formulated siRNA (from 50nM to 0.5 nM) were obtained by serial dilution of the stock solution inPBS, in order to maintain the siRNA/CADY ratio and therefore the size ofthe particle around 200 nm diameter. Mismatched siRNA and free CADY wereused as controls, and Oligofectamine (Invitrogen, USA) was used aslipid-based control delivery agent (manufacturer protocol). Theinhibition of cell proliferation was determined after 7 days.

FIG. 9: intratumoral injection of anti-cyclin B1/CADY complexes Theeffect of the anti-cyclin B1 siRNA was investigated on a tumor animalmodel, using Swiss nude mice, injected subcutaneously with 2×10⁷ 15PC3cells, A549 cells (human bronchoalveolar carcinoma) or HER2 cells.Different CADY/siRNA complexes were injected directly into the tumor(100 μl) at day 7 and at day 15. siRNA/CADY complexes were obtained asdescribed before at a ratio 1:20. The size of the tumor was measuredevery 2 days and the level of cyclin B1 protein was determined at Day 20by Western blot analysis. Results correspond to the average of 5different animals.

FIG. 10: Comparison of CADY versus CADY-S

The efficiencies of CADY-1 and CADY-S vectors for delivering anti-cyclinB1 siRNA were compared on Hela/HS68 cells, using the same assay asdescribed in the legend of FIG. 7A. The figure shows the resultsobtained on HS68 fibroblasts with a CADY-1 vector devoid of cholesterol.

FIG. 11: CADY-S-mediated delivery of anti-cyclin B 1 siRNA throughintravenous injection to block tumor growth in vivo

Experiments were performed on two tumor mouse models.

(A) 1×10⁶ 5PC3 adenocarcinoma cells were injected subcutaneously intothe flanks of NCR nude mice (day 1). siRNA/CADY-S complexes wereobtained as described before at a ratio 1:20. CADY-S/siRNA complexeswere injected intravenously at day 7. The size of the tumor was measuredevery day. Results correspond to the average of 5 different animals.

(B) 1×10⁶ tumor cells expressing luciferase were injectedintraperitoneally in the mice (4 to 8 week-old immune competent A/Jfemale mices). Tumors were monitored using in vivo Imaging System(IVIS-Xenogen), which quantified total body bioluminescence afterinjection of D-luciferin. The tumor cells are visualized on three mice,each representative of one group (siRNA alone, CADY/siRNA complexes, andCADY-S/siRNA complexes).

FIG. 12 : The different p27-derived peptides were associated with CADY-2at a molar ratio of 1/20, since it was determined that at this ratio,the average size of the particle is of about 100-200 nm in diameter. Theformulations were obtained in phosphate buffer, using a fixedconcentration of peptide of 1 μM. Cell lines were cultured in Dulbecco'sModified Eagle's Medium supplemented with 2 mM glutamine, 1% antibiotics(streptomycin 10′000 pg/ml, penicillin, 10′000 IU/ ml) and 10% (w/v)fetal calf serum (FCS), at 37° C. in a humidified atmosphere containing5% CO₂. Cells were synchronized by serum privation during 40 hrs, andcultured in Dulbecco's Modified Eagle's Medium supplemented with 2 mMglutamine, 1% antibiotics. G1 synchronized cells were released uponserum addition, then overlaid with preformed CADY-2/peptide complexes.After 30 min incubation at 37° C., 1 ml of fresh DMEM supplemented with10% fetal calf serum was added directly to the cells, without removingthe overlay of CADY/peptide complexes, and cells were returned to theincubator. The level of cell arrest in G1 was analyzed by FACS 24 hrsafter release.

(A) Protocol

(B) Results obtained with Jurkat, WI-38, HS-68 and HeLa cells.

FIG. 13 : Effect of p1p27/CADY complexes on tumor growth in vivo

The tumor animal model consists of Swiss nude mice, injectedsubcutaneously with 2×10⁷ Human H1299 adenocarcinoma cells.CADY2/p1p27complex and free CADY-2 and peptides were injected directlyinto the tumor (100-300 μg) after 1 and 4 weeks. p1p27/CADY complexeswere obtained as described before at a ratio 1/20. Controls wereperformed by injecting PBS, CADY alone, or naked p1p27 instead of thep1p27/CADY-2 complex. The size of the tumor volume was measured withcalipers once a week. Results correspond to the average of 5 differentanimals.

EXAMPLES Example 1: Peptide Synthesis

Several peptides were synthesized, and selected for their ability todeliver different kinds of cargoes, especially siRNA (CADY-1 family) andpeptides or proteins (CADY-2 family).

CADY-1 peptides have been designed so that charges are well distributedall over the vector. They have been selected for their ability todeliver an anti-GAPDH siRNA into HeLa or HS-68 cultured cells, with anefficiency sufficient to obtain at least a 50% decrease of GAPDH proteinlevel after 24 hrs of incubation with 50 nM siRNA. The efficiency ofexpression inhibition was determined using CADY-1 (SEQ ID No: 2) as areference, since this peptide enables a 95% inhibition.

An important parameter for designing CADY-2 peptides is the presence oftryptophane residues, and their repartition in the peptide. The CPPshave been selected using the p1p27 peptide (described below) as testcargo. The delivery efficiency was functionally determined by measuringthe Gi arrest of synchronized HeLa or HS68 cells, after 24 hrs ofincubation in the presence of 200 nM p1p27 peptide. The CADY-2 referencepeptide is CADY-2 (SEQ ID No: 8), which leads to 75% of cells arrestedin G1.

CADY-1 vectors for siRNA delivery CADY-1: GLWRALWRLLRSLWRLLWKA-Cya (SEQID No: 2) CADY-1b: GLWRALWRALWRSLWKLKRKV-Cya (SEQ ID No: 3) CADY-1c:GLWRALWRALRSLWKLKRKV-Cya (SEQ ID No: 4) CADY-1d:GLWRALWRGLRSLWKLKRKV-Cya (SEQ ID No: 5) CADY-1e:GLWRALWRGLRSLWKKKRKV-Cya (SEQ ID No: 6) CADY-1S:Cholesterol-GLWRALWRLLRSLWRLLWKA (SEQ ID No: 7) CADY-1S1:Cholesterol-CA_(β)-GLWRALWRLLRSLWRLL (SEQ ID No: 13) WKA CADY-1S2:GLWRALWRLLRSLWRLLWKA-Cholesterol (SEQ ID No: 7) CADY-2 vector forpeptide/protein delivery CADY-2: GLWRALWRALWRSLWKLKWKV-Cya (SEQ ID No:8) CADY-2b: GLWRALWRALWRSLWKSKRKV-Cya (SEQ ID No: 9) CADY-2c:GLWRALWRALWRSLWKKKRKV-Cya (SEQ ID No: 10) CADY-2d/1b:GLWRALWRALWRSLWKLKRKV-Cya (SEQ ID No: 3) CADY-2e:GLWRALWRLLRSLWRLLWSQ-PKKKRKV-Cya (SEQ ID No: 12)

The N-terminal extremity of these peptides is linked to an acetyl groupwhen no further indication appears. It can also be linked to acholesterol molecule (when indicated), which is either linked directlyto the N-terminus, or linked to the vector through a CA_(β) bridge,which changes the relative orientation of the cholesterol and the coreof the vector (wherein the “core of the vector” designates the peptidicpart of the vector of SEQ ID No:1).

The C-terminal extremity is linked either to a cholesterol molecule(CADY-S2) or to a cysteamide group (i.e., CO—NH—CH2—CH2—SH).

Peptide synthesis was carried out at a 0.2 mmol scale, starting from aFmoc-PAL-PEG-PS resin, using a Fmoc-continuous flow peptide synthesisinstrument (Pioneer™, Applied Biosystems, Foster City, Calif.). Thecoupling reactions were performed with 0.5 M of HATU(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) in the presence of 1 M of DIEA(diisopropylethylamine). Recoupling was carried out at positions: 6, 7,10, 11, 13, 14, 17 and 18. Protecting group removal and final cleavagefrom the resin were carried out with TFA (trifuoroacetic acid)/EDT(ethanedithiol)/thioanisole/phenol/H₂O (82.5/2.5/5/5/5%) during 5 h 30min. The reaction mixture was then filtered through a cotton filter incold diethyl ether. Since there occurred no peptide precipitation, etherwas removed under rotary evaporation to obtain a yellow oil. Neat etherwas added to the oil and stored at −20° C. overnight to allow thepeptide to precipitate. A certain quantity of peptide has alreadyprecipitated in the cleavage cocktail (it is unusual for a peptide toprecipitate in a near pure solution of TFA). This precipitate (+thecotton and the resin) was resuspended in CH₃CN (containing 0.08% TFA)and H₂O. After filtration and thorough washes by CH₃CN/TFA and H₂O, thefiltrate was lyophilized prior to purification.

The crude peptide was purified by RP-HPLC on a C18 column (InterchromUP5 WOD/25M Uptispere 300 5 ODB, 250×21.2 mm). Electrospray ionizationmass spectrum was in complete agreement with the proposed structure.

Example 2: Structure of CADY When Interacting with a Membrane

The structure of CADY peptides was investigated by spectroscopy. It wasdemonstrated by circular dichroism that CADY adopts a helical structurein water or in phosphate solution. CADY forms a secondary amphipathicorganization. Helical structure of CADY results in a hydrophobic and ahydrophilic faces. Molecular dynamic analysis reveals that CADY formsmultimeric structures, involving at least 4 CADY molecules within themembrane (FIGS. 1 and 2). FTIR measurement of transferred CADY/membranehave confirmed that CADY peptides form an helical structure in themembrane.

Example 3: CADY Forms Stable Particles with Cargoes Through Non CovalentInteractions

The inventors have investigated the formation of CADY/cargo complexesand demonstrated that CADY peptides are able to form stable complexeswith their cargoes. CADY-1 interact mainly with siRNA (FIG. 3A) andCADY-2 mainly with peptides and proteins (FIG. 3B). Binding of cargoesto CADY was monitored by steady state fluorescence spectroscopy, usingthe intrinsic fluorescence associated to the Trp residue of CADY. Foreach CADY peptide, a fixed concentration of CADY was titrated byincreasing concentration of cargoes. The binding of the cargo induced amarked quenching of fluorescence of both CADY peptides, and thedissociation constants were calculated from data fitting, using aquadratic equation which allows the determination of the ratioCADY/cargo. Dissociation constants and ratios were estimated to 2 nM forCADY-1/siRNA and 19 nM for CADY2/peptide, with a ratio at saturation of1/10 and 1/20, respectively, whatever the variant of CADY-1 or CADY-2.However, cholesterol modification (i.e., addition) reduced 2 to 10 foldsthe affinity of CADY-1 for the siRNA. These results suggest that in bothcases, more than one peptide interact with the cargo, and that bothCADY-1 and CADY-2 are able to form particles with their respectivecargoes.

Example 4: CADY Forms Stable and Non-Toxic Particles

The toxicity of CADY peptides has been investigated on different celllines. Cells were incubated in the presence of increasing concentrationsof CADY (from 0.1 μM to 1 mM) for 12 hrs and cell viability wasestimated using MTT assay. Data reported in FIG. 4A demonstrated thatCADY-1 is not toxic on the tested cell lines, up to a concentration of100 μM, with a EC₅₀ of 0.5 mM.

The different CADY variants have been tested on HS-68 and Hela cells.The results are reported in the Table 1 below.

TABLE 1 free complexed Peptides EC50 (mM) EC50 (mM) CADY-1 0.4 >2CADY-1b 0.5 >2 CADY-1c 0.45 >2 CADY-1d 0.5 >2 CADY-1e 0.34 1.5 CADY-S0.2 1 CADY-S2 0.25 0.8 CADY-2 0.23 1 CADY-2b 0.28 0.9 CADY-2c 0.34 0.8CADY-2d 0.38 1.5 CADY-2e 0.43 >2 KALA 0.07 GALA 0.05 TAT 0.1Lipofectamine 0.2

The inventors demonstrated that association with cargo molecule (ratio1/20) significantly reduced the toxicity of the carrier, an average EC₅₀of 2 mM was calculated. The lack of toxicity of CADY/cargo complexes canbe directly associated to absence of free carrier peptide in thesolution (FIG. 4B). Lipofectamine and other membranolytic CPP (KALA,GALA, TAT), which exhibit more that 70% toxicity at concentration of 100μM, were used as controls.

Example 5: CADY-Mediated Delivery of siRNA

The ability of CADY-1 and variants to deliver siRNA was evaluated onboth adherent and suspension cell lines, using GAPDH as the target ofsiRNA.

siRNA targeting the 3′UTR of GAPDH were from the Silencer™ GAPDH siRNAkit (Ambion). Fluorescent labeling of siRNA was performed using eitherthe Fam or the Cy3 Silencer™ labeling kit (Ambion) and modified asdescribed in the manufacturer's protocol. pRL-Luc reporter gene was fromPromega and siRNA targeting luciferase sense 5′-CUUACGCUGAGUACUUCGATT-3′(SEQ ID No: 14) and antisense 3′-TTGAAUGCGACUCAUGAAGCU-5′ (SEQ ID No:15) and mismatch sense 5′-CGUACGCGGAAUACUUCGATT-3′ (SEQ ID No: 16) andantisense 3′-TTGCAUGCGCCUUAUGAAGCU-5′ (SEQ ID No: 17) siRNA wereobtained from Genset Oligos.

The results, presented in FIG. 5, show that CADY-1 is more efficientthan the lipofectamine control. The percentage of GAPDH knockdown,estimated by comparison with a control essays using a mismatch siRNAdelivered with CADY at the same molecular ratio, is also reported inTable 2 below. Data are the average of 4 separated experiments.

TABLE 2 Cell lines Knockdown Hela 85% Jurkat 80% HepG2 70% C2C12 70% MEF70% HS-68 80% CEM-SS 80% U2OS 75% MCF7 70% MT4 65% HER2 60% MDA-MB 75%Balb/c3T3 80%

Example 6: Mechanism of Cellular Uptake of CADY-1

Whatever the CPP, the common major concern in the development of new PTDstrategy is to avoid any endosomal pathway and/or to facilitate theescape of the cargoes from the early endosomes in order to limit theirdegradation. The cellular uptake mechanism of CADY-1 was thereforeinvestigated in detail. The inventors demonstrated that, in contrast tomost of the delivery systems described so far for siRNA, CADY-mediateddelivery of active siRNA is independent of the endosomal pathway anddirectly correlated to the size of the particle CADY/siRNA.

Indeed, results reported in FIG. 6A revealed that efficiency of CADY isdependent on the size of the particle formed with siRNA. Optimalbiological response was obtained for a CADY/siRNA ratio of 1:20 to 1:25,which corresponds to a nano particle (size of 100-200 nm). Therefore,calibration of the formulation is an essential parameter in theefficiency of CADY-1.

Results reported in FIG. 6B demonstrated that none of the testedinhibitors affected the cellular uptake of CADY and the associated siRNAbiological response. In contrast to other CPP, the mechanism of cellularuptake of CADY/siRNA particle is independent of the endosomal pathway.However, the size of the particle and CADY/siRNA molar ratio need to becontrolled, so that the diameter of the particles remains below 200 nmand the CADY/siRNA molar ratio remains under 1:25. In contrast, forparticles of diameter>500 nm, the mechanism is partially associated tothe endosomal pathway.

These data demonstrate that calibration of the formulation is anessential parameter in the efficiency of CADY-1, and that the optimalformulation for CADY/siRNA is: ratio 1:20 to 1:25 and nano particle(size of 100-200 nm).

Example 7: CADY-Mediated In Vitro Delivery of siRNA Targeting Cyclin-B1

Cell cycle progression is driven by sequential activation of essentialheterodimeric protein kinase complexes (Cdk/cyclin complexes). Most ofthe drugs currently designed to target cell cycle progression aredirected against the kinase activity of the Cdk subunits. However, assuch drugs generally tend to affect other cellular kinases nonspecifically, the inventors have decided to target the regulatory cyclinsubunit instead, as a more appropriate means of improving both theselectivity and the efficiency of cell cycle inhibitors. In particular,cyclin B1 constitutes a key target for cancer therapy, as a component ofthe essential “Mitosis Promoting Factor”, together with protein kinaseCdk1.

A siRNA molecule was hence designed to target cyclin B 1. The followingsiRNA sequence was selected out of 10 different sequences, and theoligonucleotides were obtained from proligo:

Si-Cyclin B sense: 5′-GGCGAAGAUCAACAUGGCATT-3′ (SEQ ID No: 18) Si-CyclinB antisense: 5′-UGCCAUGUUGAUCUUCGCCTT-3′ (SEQ ID No: 19) Mismatch sense:5′-GGUGAAGAUCAGCAUGGCATT-3′ (SEQ ID No: 20) Mismatch antisense:5′UGCCAUGUCGAUCUUCACCTT3′ (SEQ ID No: 21)

The potency of this siRNA was investigated using CADY as deliverysystem. The biological response of CADY-mediated delivery of siRNAtargeting cyclin B 1 was examined. To this aim, the inventors firstassessed whether cyclin B1 protein levels were downregulated byCADY-mediated delivery of siRNA, and to what extent, compared todelivery of siRNA using classical lipid formulation.

As shown in FIG. 7A, delivery of siRNA cyclin B 1 is associated to acell cycle arrest in G2, characteristic of a knock down of the cyclin B1protein. Western blot analysis confirmed that the observed G2-arrest isdirectly associated to a dramatic knock down of the cyclin B1 proteinlevel (FIG. 7B). Interestingly, the G2 arrest is observed at lowconcentration of siRNA of 2 nM. This demonstrates the high potency ofCADY for siRNA delivery and rapid release into the cytoplasm. UsingCADY-mediated delivery can hence enable a significant response with onlylow concentrations of siRNA, which will limit problems related tounspecific targeting and toxicity.

Example 8: Anti-cyclin-B1 siRNA/CADY Inhibits Proliferation of CancerCells

The potency of CADY/siRNA complex to block cancer cell proliferation wasthen investigated.

As shown on FIG. 8, the anti-cyclin B1 siRNA significantly blocks cancercell proliferation when associated to CADY. 40% and 72% of inhibitionwere obtained with siRNA concentrations of 0.1 nM and 1 nM,respectively, associated to CADY-1, whereas the same concentrations werenot efficient when lipofectamine was used as vector. These results arein perfect agreement with those obtained on other cell lines and confirmthe high potency of CADY and the possibility of using very lowconcentration (sub nanomolar) of siRNA.

These data are the first demonstration that siRNA can work atconcentrations lower that 1 nM. Such low concentrations of siRNA induceda strong biological response, suggesting that CADY-mediated delivery ishighly efficient. This is certainly correlated with the rapid release ofthe siRNA in the cytoplasm, independently of the endosomal pathway.

Example 9: Anti-Cyclin-B1 siRNA/CADY Inhibits Tumor Growth: IntratumoralInjection

The effect of the anti-cyclin B1 siRNA/CADY complexes was theninvestigated on a tumor animal model, to determine to what extend theresults obtained on the proliferation of cancer cells could beextrapolated to the inhibition of tumor growth in vivo.

The results are shown on FIG. 9. In a control experiment, the inventorsobserved that 20 days after injection of PBS, CADY or naked siRNA, thetumor size increased by 2,5 fold. In contrast, no growth of the tumorwas observed, when injected with CADY/siRNA even with low concentrationof siRNA (1 μg).

These results suggested that siRNA specifically inhibits tumor growthand, thanks to the used of CADY as carrier, only a low concentration ofsiRNA is required for a marked anti-tumoral effect. Moreover, asignificant reduction in the tumor size was observed after a secondinjection of CADY/siRNA. Western blot analysis showed that the level ofcyclin B 1 was dramatically reduced.

These results demonstrate that CADY constitutes an excellent tool for invivo delivery of siRNA.

Example 10: Cholesterol Modified-CADY-Formulation for In VivoAdministration

The above results demonstrate that CADY-1 constitutes an excellent toolfor in vivo application of siRNA. However, although CADY-1/siRNAformulation is highly efficient upon intra-tumoral injection, theobserved response to siRNA was very limited when the complexes wereinjected intravenously or via the intra-peritoneal route.

A new variant of CADY was hence designed to obtain nano-particlesincluding siRNA, which are stable in vivo and administrable throughintravenous injections. In that purpose, a cholesterol group was addedat the N- terminus (CADY-S) or at the C-terminus (CADY-S2) of CADY.Cholesterol linking at the C-terminus was performed through a disulfidebond with the cysteamide group. Linking at the N-terminus was performedthrough the activation of the amino group and formation of a disulfidebond.

The ability of both CADY-S and CADY-S2 to deliver siRNA was evaluatedusing anti-cyclin B 1 siRNA. In order to identify the optimalformulation, different molar ratios of CADY were used in complex with 50nM siRNA. Formulations were ranked based on the G2 arrest associated toknock down of cyclin B 1.

The results, presented in FIG. 10, show that the presence of cholesterolattached to CADY does not affect its efficiency to deliver siRNA incultured cells. The optimal formulation is similar to that obtained withCADY. The optimal size for the particle is of about 200 nm diameter.

The in vivo efficiency of CADY-S was then investigated, in order todetermine if the cholesterol-modified form of CADY can improve siRNAdelivery through intravenous injection and block tumor growth in vivo.Experiments were performed on two tumor mouse models, in which 5PC3 orHuman H1299 adenocarcinoma cells were injected subcutaneously into theflanks of NCR nude mice.

The results of both experiments demonstrate that cholesterol-modifiedCADY was able to deliver siRNA in vivo, and induced a significantbiological response by blocking tumor growth (FIG. 11). The presence ofcholesterol improves the stability of the particle after intravenousinjection. Moreover, only one injection at low concentration of siRNA(10 μg) of siRNA was required to fully abolish the tumor growth.

These results constitute the first in vivo therapeutic application of acarrier system/siRNA complex using physiological doses. CADY henceappears as the best vector for systematic in vivo delivery of siRNA,since other potent methods (such as cationicpolymer/cholesterol-modified siRNA) required an everyday IV injection ofsiRNA, at concentrations 10 fold higher than those used in theabove-described experiments, to obtain a similar efficiency.

Example 11: CADY-Mediated Delivery of Therapeutic Peptides

Protein transduction technology described so far requires the attachmentof the Protein Transduction Domain (PTD) to the target peptide orprotein, which can be achieved either by chemical cross-linking or bycloning and expression of a fusion protein. Conjugate method offersseveral advantages for rationalization and control of the stoechiometry,as well as for reproducibility and calibration of the PTD-cargo for invivo application. However, this approach is limited from a technicalpoint of view and by a risk of alteration of the biological activity ofthe cargoes.

CADY vectors, especially CADY-2 vectors, offer an alternative to thecovalent PTDs technology, for the delivery of full-length proteins andpeptides into mammalian cells.

In higher eukaryotes, the cell cycle progression is coordinated byseveral closely related Ser/Thr protein kinases, resulting fromassociation of a catalytic Cyclin-dependent kinase (CDK) subunit with aregulatory cyclin subunit. CDK/cyclin complexes are regulated by bothphosphorylation/dephosphorylation and protein-protein interactions withdifferent partners, including structural CDK inhibitors (CKI). Geneticevidence supports a strong correlation between alterations in theregulation of CDKs and the molecular pathology of cancer. Thus, in thepast few years, there has been considerable interest in the developmentof inhibitors of CDK protein kinases. A greater understanding of thecell cycle and of the inherent complexity of CDK regulation now offers anumber of possible routes to their inhibition (Swanton 2004). One of thecurrently most developed strategies to inhibit CDKs is based on thedesign of small molecules that target the ATP-binding site, therebyinterfering directly with their catalytic activity.

The tumor suppressor protein p27 is essential in the control of the G1/Stransition. Alteration in p27 regulation results in aberrations in cellcycle progression and development of tumors. p27 protein interacts withthe Cdk/cyclin complex in order to maintain the complex in an inactiveconformation which controls the rate of the G1 phase. The molecularmechanism by which p27 induces cell cycle arrest was investigated indetails, and peptide motifs which block both cell cycle in G1 and cancercell proliferation have been identified.

Based on the X-ray structure of the cdk2/cyclinA/p27 complex (Jeffrey,Russo et al. 1995), the inventors have selected three peptides:

p1p27 RVSNGSPSLERMDARQAEHPKPSACRNL (SEQ ID No: 22) pRXL PSLERMDAR, (SEQID No: 23) and p2p27 NRTEENYSDGSPNAGSVEQTPKKPGLR (SEQ ID No: 24) as anegative control.

Peptides were synthesized and purified as described previously (Mery,Granier et al. 1993; Morris, Vidal et al. 1997), then their ability toalter the cell cycle progression was investigated.

As shown on FIG. 12, the delivery of the peptides p1p27 or pRXL induceda cell cycle arrest in G1 in all tested cell lines. In contrast, noeffect was observed with CADY or free p1p27, which suggested that CADYdramatically improves the cellular uptake of the peptide, without anytoxicity associated to the carrier. Moreover, the fact that no effectwas observed with the control peptide p2p27 demonstrates the highspecificity of the peptide. In comparison to results previouslypublished with pRXL using a TAT or Pen covalent based strategy(Nagahara, Vocero-Akbani et al. 1998; Chen, Sharma et al. 1999; Snyder,Meade et al. 2003), the efficiency with CADY strategy is at least 2-foldbetter.

Example 12: CADY2-Mediated In Vivo Delivery of p27 Inhibits Tumor Growth

The inventors then investigated the ability of the p1p27/CADY complexesto block tumor growth in vivo.

The results are shown on FIG. 13.

In control experiments, in which PBS, CADY alone or naked p1p27 wereinjected, the tumor size had increased by 5-fold, 5 weeks afterinjection. In contrast, no growth of the tumor was observed, wheninjected with CADY/p1p27. These results suggest that p1p27 specificallyinhibits tumor growth, and that the use of CADY as carrier enables toadminister only a low amount of p1p27 for a marked anti-tumoral effect.Moreover, a significant reduction in the tumor size was observed after asecond injection of CADY/p1p27. These results show that CADY constitutesan excellent tool for in vivo application of therapeutic peptides.

Other experiments, performed with peptides having an anti-viral or anantitumoral activity, as described in (Morris, Robert-Hebmann et al.1999) or in (Gondeau, Gerbal-Chaloin et al. 2005), demonstrated thatCADY-2 peptides improve the delivery of several distinct proteins andpeptides into different cell lines in a fully biologically active form,without the need for prior chemical covalent coupling or denaturationstep. CADY technology hence constitutes a powerful tool for basicresearch, and demonstrated to be extremely powerful for studying therole of proteins, and for targeting specific protein/proteininteractions in vitro as well as in vivo.

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1-23. (canceled)
 24. A cell-penetrating peptide with amino acid sequenceselected from the group consisting of GLWRALWRLLRSLWRLLWKA (SEQ ID No:2); GLWRALWRALWRSLWKLKRKV (SEQ ID No: 3); GLWRALWRALRSLWKLKRKV (SEQ IDNo: 4); GLWRALWRGLRSLWKLKRKV (SEQ ID No: 5); GLWRALWRGLRSLWKKKRKV (SEQID No: 6); GLWRALWRLLRSLWRLLWKA (SEQ ID No: 7); GLWRALWRALWRSLWKLKWKV(SEQ ID No: 8); GLWPALWRALWRSLWKSKRKV (SEQ ID No: 9);GLWPALWRALWRSLWKKKRKV (SEQ ID No: 10); and GLWRALWRLLRSLWRLLWSQ (SEQ IDNo: 11).
 25. The cell-penetrating peptide of claim 24, furthercomprising, covalently linked to the C-terminal end of said amino acidsequence, one or several groups chosen amongst a cysteamide, a cysteine,a thiol, an amide, a carboxyl, a linear or ramified C1-C6 alkyloptionally substituted, a primary or secondary amine, an osidicderivative, a lipid, a phospholipid, a fatty acid, a cholesterol, apoly-ethylene glycol, a nuclear localization signal, and/or a targetingmolecule.
 26. The cell-penetrating peptide of claim 24, furthercomprising, covalently linked to the N-terminal end of said amino acidsequence, one or several chemical entities chosen amongst an acetyl, afatty acid, a cholesterol, a poly-ethylene glycol, a nuclearlocalization signal, and/or a targeting molecule.
 27. A complexcomprising a cell-penetrating peptide according to claim 24 and a cargoselected amongst nucleic acids, peptides, proteins, contrast agents, andtoxins.
 28. The complex of claim 27, wherein said cargo is a siRNAselected to silence a target mRNA.
 29. The complex of claim 28, whereinthe amino acid sequence of the cell-penetrating peptide is chosen in thegroup consisting of GLWRALWRLLRSLWRLLWKA; (SEQ ID No: 2)GLWRALWRALWRSLWKLKRKV; (SEQ ID No: 3) GLWRALWRALRSLWKLKRKV; (SEQ ID No:4) GLWRALWRGLRSLWKLKRKV; (SEQ ID No: 5) GLWRALWRGLRSLWKKKRKV; (SEQ IDNo: 6) and GLWRALWRGLRSLWKKKRKV. (SEQ ID No: 7)


30. The complex of claim 27, wherein said cargo is a peptide or aprotein.
 31. The complex of claim 31, wherein the amino acid sequence ofthe cell-penetrating peptide is chosen in the group consisting ofGLWRALWRALWRSLWKLKWKV; (SEQ ID No: 8) GLWRALWRALWRSLWKSKRKV; (SEQ ID No:9) GLWRALWRALWRSLWKKKRKV; (SEQ ID No: 10) GLWRALWRALWRSLWKLKRKV; (SEQ IDNo: 3) GLWRALWRLLRSLWRLLWSQ (SEQ ID No: 11) andGLWRALWRLLRSLWRLLWSQPKKKRKV. (SEQ ID No: 12)


32. The complex of claim 27, wherein the cell-penetrating peptidecomprises an acetyl group covalently linked to its N-terminus, and/or acysteamide group covalently linked to its C-terminus.
 33. The complex ofclaim 27, wherein the cell-penetrating peptide further comprises acholesterol molecule, covalently linked to its C-terminus or itsN-terminus.
 34. The complex of claim 27, wherein the size of the complexis between 50 and 300 nm.
 35. The complex of claim 34, wherein the sizeof the complex is between 100 and 200 nm.
 36. The complex of claim 27,wherein at least part of the cell-penetrating peptides are bound to atargeting molecule.
 37. A therapeutic composition comprising the complexaccording to claim
 27. 38. A method for delivering a molecule into acell in vitro, comprising putting said cell into contact with a complexcomprising said molecule and the cell-penetrating peptides according toclaim
 27. 39. A method for treating cancer, comprising a step ofadministering to a patient in need thereof a composition comprising acomplex according to claim
 27. 40. The method of claim 39, wherein saidcargo comprises a cell-penetrating peptide chosen in the groupconsisting of CADY-1 (Ac-GLWRALWRLLRSLWRLLWKA-Cya) and CADY-2(Ac-GLWRALWRALWRSLWKLKWKV-Cya).
 41. The method of claim 40, wherein saidtherapeutic composition comprises anti-cyclin B 1 siRNA/CADY complexes.42. The method of claim 40, wherein said therapeutic compositioncomprises p1p27/CADY complexes and/or pRXL/CADY complexes.